Anti-coronavirus vaccines

ABSTRACT

The invention provides stable coronavirus spike proteins. Immunogenic compositions comprising same and the methods of using these immunogenic compositions are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Nos.63/064,225, filed Aug. 11, 2020, 63/088,708, filed Oct. 7, 2020 and63/145,045, filed Feb. 3, 2021 the entire contents of which areincorporated herein by reference in their entirety.

FIELD OF THE INVENTION

This invention is in the field of recombinant coronavirus Spike proteinsand immunogenic compositions containing same.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted via EFS-Web as ASCII compliant text file format (.txt), and ishereby incorporated by reference in its entirety. The ASCII file wascreated on Oct. 7, 2020, is named “Sequence_Listing_000360_ST25” and hassize of 37,849 bytes. This Sequence Listing serves as paper copy of theSequence Listing required by 37 C.F.R. § 1.821(c) and the SequenceListing in computer-readable form (CRF) required by 37 C.F.R. §1.821(e). A statement under 37 C.F.R. § 1.821(f) is not necessary.

BACKGROUND

Coronaviruses are a large family of viruses that can cause illnessesranging widely in severity. The first known severe illness caused by acoronavirus emerged with the 2003 Severe Acute Respiratory Syndrome(SARS) epidemic in China. A second outbreak of severe illness began in2012 in Saudi Arabia with the Middle East Respiratory Syndrome (MERS).

On December 31 of 2019, Chinese authorities alerted the World HealthOrganization of an outbreak of a novel strain of coronavirus causingsevere illness, which was subsequently named SARS-CoV-2. SARS-CoV-2 isthe virus that causes the disease referred to as COVID-19. As of Jul.16, 2020, nearly 13.6 million COVID-19 cases have been documentedworldwide, although many more mild cases have likely gone undiagnosed.The virus has killed over 585,000 people.

Shortly after the epidemic began, Chinese scientists sequenced thegenome of SARS-CoV-2 and made the data available to researchersworldwide. The number of COVID-19 cases have been increasing because ofhuman to human transmission after a single introduction into the humanpopulation.

SARS-CoV-2 spike proteins are located on the outside of the virus. Thevirus uses its spike protein to grab and penetrate the outer walls ofhuman and animal cells. Scientists have focused on two distinctivefeatures of SARS-CoV-2′s spike protein—the Receptor Binding Domain (RBD)portion that binds to cells and the cleavage site that opens the virusup and allows it to enter host cells. The S1 and S2 subunits of thespike protein are responsible for receptor recognition and membranefusion, respectively.

Scientists are still learning about this virus, but it appears that itcan spread from people to animals in some situations, especially afterclose contact with a person sick with COVID-19.

Based on information available on the website of the Centers for DiseaseControl and Prevention (CDC), updated Jun. 22, 2020, we know that cats,dogs, and a few other types of animals can be infected with SARS-CoV-2,but we do not yet know all of the animals that can get infected. Therehave been reports of animals being infected with the virus worldwide.

A small number of pet cats and dogs have been reported to be infectedwith the virus in several countries, including the United States. Mostof these pets became sick after contact with people with COVID-19.Several lions and tigers at a New York zoo tested positive forSARS-CoV-2 after showing signs of respiratory illness. Public healthofficials believe these large cats became sick after being exposed to azoo employee who was infected with SARS-CoV-2.

SARS-CoV-2 was recently discovered in mink (which are closely related toferrets) on multiple farms in the Netherlands. The mink showedrespiratory and gastrointestinal signs; the farms also experienced anincrease in mink deaths. Because some workers on these farms hadsymptoms of COVID-19, it is likely that infected farm workers were thesource of the mink infections. Some farm cats on several mink farms alsodeveloped antibodies to this virus, suggesting they had been exposed tothe virus at some point.

The CDC, U.S. Department of Agriculture (USDA), and state public healthand animal health officials are working in some states to conduct activesurveillance of SARS-CoV-2 in pets, including cats, dogs, and othersmall mammals, that had contact with a person with COVID-19. Theseanimals are being tested for SARS-CoV-2 infection, as well as tested tosee whether the pet develops antibodies to this virus. This work isbeing done to help us better understand how common SARS-CoV-2 infectionmight be in pets as well as the possible role of pets in the spread ofthis virus. The USDA maintains a list of cases of SAR-CoV-2 (the samevirus that causes COVID-19 in humans) in animals in the United Statesthat have been confirmed by the USDA's National Veterinary ServicesLaboratories.

The development of human vaccines against SARS-CoV-2 is underway but aveterinary vaccine is also needed.

SUMMARY OF INVENTION

In the first aspect, the invention provides a composition comprising acoronavirus, a Spike protein of said coronavirus or an immunogenicfragment of said Spike protein, and an adjuvant comprising a saponin, asterol, and a CpG-containing immunostimulatory oligonucleotide. Incertain embodiments of this first aspect, the adjuvant consistsessentially or consists of the saponin, the sterol, and theCpG-containing immunostimulatory oligonucleotide. In any of theembodiments described above, the saponin may be a triterpenoid saponins,preferably extracted from bark of Quillaia Saponaria, and the sterol maybe selected from the group consisting of β-sitosterol, stigmasterol,ergosterol, ergocalciferol, and cholesterol. In any of the embodimentsdescribed above, the saponin may be present in the amount of about 20 μgper dose and the sterol may be present in the amount of about 20 μg perdose.

In the second aspect, the invention provides a composition comprisingcoronavirus, a Spike protein from said coronavirus or an immunogenicfragment of said Spike protein, and an adjuvant comprising (orconsisting essentially of or consisting) a CpG containingimmunostimulatory oligonucleotide and a glycolipid according to FormulaI,

wherein, R¹ is hydrogen, or a saturated alkyl radical having up to 20carbon atoms; X is —CH₂—, —O— or —NH—; R² is hydrogen, or a saturated orunsaturated alkyl radical having up to 20 carbon atoms; R³, R⁴, and R⁵are independently hydrogen, —SO₄ ²⁻, —PO₄ ²⁻, —COC₁₋₁₀ alkyl; R⁶ isL-alanyl, L-alpha-aminobutyl, L-arginyl, L-asparginyl, L-aspartyl,L-cysteinyl, L-glutamyl, L-glycyl, L-histidyl, L-hydroxyprolyl,L-isoleucyl, L-leucyl, L-lysyl, L-methionyl, L-ornithinyl, L-phenyalany,L-prolyl, L-seryl, L-threonyl, L-tyrosyl, L-tryptophanyl, and L-valyl ortheir D-isomers.

In certain embodiments of this second aspect, the glycolipid isN-(2-Deoxy-2-L-leucylamino-β-D-glucopyranosyl)-N-octadecyldodecanoylamideor a salt thereof, such as an acetate thereof. In any of the embodimentsof this second aspect of the invention, the glycolipid may be present inthe amount of about 250 μg per dose.

In any of the embodiments of the first and/or the second aspect of theinvention as described above, the immunostimulatory oligonucleotide maybe a P-class immunostimulatory oligonucleotide characterized by thepresence of one or more TLR-9 activating motif (s) and two palindromesor two complementarity areas. Preferably, said P-class immunostimulatoryoligonucleotide is 5′ modified, and more preferably, wherein said Pclass immunostimulatory oligonucleotide comprises at least 22 contiguousnucleotides of SEQ ID NO: 8. In certain embodiments of the first and/orthe second aspect of the invention, the CpG containing immunostimulatoryoligonucleotide is present in the amount of about 20 to about 50 μg perdose.

In a third aspect, the invention provides a composition comprising acoronavirus, a Spike protein from said coronavirus or an immunogenicfragment of said Spike protein, and an adjuvant comprising a saponin, asterol, a quaternary ammonium compound, and a polyacrylic acid polymer.In certain embodiments of this third aspect of the invention, thesaponin is a triterpenoid saponin, preferably extracted from the bark ofQuillaia Saponaria, such as is Quil A and the sterol is selected fromthe group consisting of β-sitosterol, stigmasterol, ergosterol,ergocalciferol, and cholesterol, and the quaternary ammonium compound isDDAB. In certain embodiments, said Quil A is present in the amount ofabout 20 μg per dose, the sterol is cholesterol and is present in theamount of about 20 μg per dose, the DDAB is present in the amount ofabout 10 μg per dose, and the polyacrylic acid polymer is present in theamount of about 0.05% v/v.

In certain embodiments of the first, the second, or the third aspect ofthe invention, the coronavirus is SARS-2 coronavirus and the antigen isthe Spike protein or the immunogenic fragment thereof. In certainembodiments applicable to the first, the second and the third aspect ofthe invention, wherein the Spike protein is at least 90% identical toSEQ ID NO: 13, with a proviso that said protein is in a pre-fusionstate. In certain embodiments, the pre-fusion state is conferred bysubstitution of amino acids at position 973 and/or 974 of SEQ ID NO: 13.In certain preferred embodiments, the amino acids at positions 973 and974 are substituted to prolines.

In certain embodiments, applicable to any of the above-describedembodiments, spike protein or the fragment thereof comprises a mutationin SEQ ID NO: 15, preferably, a mutation wherein SEQ ID NO: 15 isreplaced with SEQ ID NO: 16. In further embodiments, applicable to allspike proteins or fragments thereof described above, the protein mayfurther comprise a foldon sequence such as, for example SEQ ID NO:12. Incertain embodiments, the composition according to the first, the second,or the third aspect of the invention comprises a spike protein or animmunogenic fragment thereof in the amount of about 20 μg per dose.

The fourth aspect of the invention provides a method of inducing animmune response in a subject in need thereof, the method comprisingadministering to said subject the composition according to any one theembodiments described above.

In certain embodiments of this fourth aspect, wherein said immunogeniccomposition is administered to said subject in a prime administrationand in a boost administration, wherein the boost administration isbetween about 14 and about 42 days after the prime administration.

Preferably, the immune response is a protective immune response and, incertain embodiments, said protective immune response is retained forsixth or twelve months after the prime vaccination.

In certain embodiments of this fourth aspect, said subject is a canineand the adjuvant in said immunogenic composition comprises the saponin,the sterol, and the CpG-containing immunostimulatory oligonucleotide. Inother embodiments, said subject is a feline and the adjuvant in saidimmunogenic composition comprises the CpG-containing immunostimulatoryoligonucleotide and the glycolipid according to Formula I. In yet otherembodiments, said subject is a feline and the adjuvant in saidimmunogenic composition comprises the sterol, the saponin, thequaternary ammonium compound and the polyacrylic acid polymer.

DETAILED DESCRIPTION Definitions

“About” or “approximately,” when used in connection with a measurablenumerical variable, refers to the indicated value of the variable and toall values of the variable that are within the experimental error of theindicated value (e.g., within the 95% confidence interval for the mean)or within 10 percent of the indicated value, whichever is greater. Withregard to time period, the term “about” refers to the indicated valueand to a range within 10% of the indicated value (e.g., “about 8 months”includes 8 months as well as 8 months plus or minus 10%), except theupper limit of “about 11 months” is 12 months and the upper limit of“about 12 months” is 12.5 months.

“Adjuvant” means any substance that increases the humoral or cellularimmune response to an antigen. Adjuvants are generally used toaccomplish two objectives: the controlled release of antigens from theinjection site, and the stimulation of the immune system.

“Antibody” refers to an immunoglobulin molecule that can bind to aspecific antigen as the result of an immune response to that antigen.Immunoglobulins are serum proteins composed of “light” and “heavy”polypeptide chains having “constant” and “variable” regions and aredivided into classes (e.g., IgA, IgD, IgE, IgG, and IgM) based on thecomposition of the constant regions.

“Antigen” or “immunogen” refers to any substance that is recognized bythe animal's immune system and generates an immune response. The termincludes killed, inactivated, attenuated, or modified live bacteria,viruses, or parasites. The term “antigen” also includes polynucleotides,polypeptides, recombinant proteins, synthetic peptides, protein extract,cells (including tumor cells), tissues, polysaccharides, or lipids, orfragments thereof, individually or in any combination thereof. The termantigen also includes antibodies, such as anti-idiotype antibodies orfragments thereof, and to synthetic peptide mimotopes that can mimic anantigen or antigenic determinant (epitope).

“Buffer” means a chemical system that prevents change in theconcentration of another chemical substance, e.g., proton donor andacceptor systems serve as buffers preventing marked changes in hydrogenion concentration (pH). A further example of a buffer is a solutioncontaining a mixture of a weak acid and its salt (conjugate base) or aweak base and its salt (conjugate acid).

“Conservative substitutions” refer to replacement of one amino acid withanother amino acids, wherein the replacing and the replaced amino acidhave similar structures. For example, changes which result in thesubstitution of one negatively charged residue for another, such asaspartic acid for glutamic acid, or one positively charged residue foranother, such as lysine for arginine, can also be expected to produce aprotein with substantially the same functional activity.

The following six groups each contain amino acids that are typicalconservative substitutions for one another: [1] Alanine (A), Serine (S),Threonine (T); [2] Aspartic acid (D), Glutamic acid (E); [3] Asparagine(N), Glutamine (Q); [4] Arginine (R), Lysine (K), Histidine (H); [5]Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and [6]Phenylalanine (F), Tyrosine (Y), Tryptophan (W), (see, e.g., US PatentPublication 20100291549).

“Consisting essentially” as applied to the adjuvant formulations refersto formulation which does not contain unrecited additional adjuvantingor immunomodulating agents in the amounts at which said agent exertmeasurable adjuvanting or immunomodulating effects. Preferably, ifpresent, such unrecited additional adjuvanting or immunomodulatingagents are in the amount that is below detection threshold.

“Dose” refers to a vaccine or immunogenic composition given to a subjectin a single administration

“Immune response” in a subject refers to the development of a humoralimmune response, a cellular immune response, or a humoral and a cellularimmune response to an antigen. Immune responses can usually bedetermined using standard immunoassays, cell-based assays, andneutralization assays, which are known in the art.

“Immunologically effective amount” or “effective amount to produce animmune response” of an antigen is an amount effective to induce animmunogenic response in the recipient. The immunogenic response may besufficient for diagnostic purposes or other testing or may be adequateto prevent signs or symptoms of disease, including adverse healtheffects or complications thereof, caused by infection with a diseaseagent. Either humoral immunity or cell-mediated immunity or both may beinduced. The immunogenic response of an animal to an immunogeniccomposition may be evaluated, e.g., indirectly through measurement ofantibody titers, cytokine assays, lymphocyte proliferation assays, ordirectly through monitoring signs and symptoms after challenge with wildtype strain, whereas the protective immunity conferred by a vaccine canbe evaluated by measuring, e.g., reduction in clinical signs such asmortality, morbidity, temperature number, overall physical condition,and overall health and performance of the subject. The immune responsemay comprise, without limitation, induction of cellular and/or humoralimmunity.

“Immunogenic” means evoking an immune or antigenic response. Thus, animmunogenic composition would be any composition that induces an immuneresponse.

“Pharmaceutically acceptable” refers to substances, which are within thescope of sound medical judgment, suitable for use in contact with thetissues of subjects without undue toxicity, irritation, allergicresponse, and the like, commensurate with a reasonable benefit-to-riskratio, and effective for their intended use.

The term “protective immune response” refers to the immune response,elicited by an immunogenic composition or a vaccine in a subject,wherein upon the challenge with the coronavirus against which the animalwas immunized, the subject does not get infected (complete protection)or exhibits symptoms of lesser magnitude and/or duration compared to thenon-immunized animal (partial protection). In a particularly preferredembodiment of partial protection, the immunized and challenged subjectdoes not shed the coronavirus, or the magnitude and/or the duration ofshedding is decreased. Thus, protective immune response prevents theinfection and/or lessens the symptoms and/or the duration of theinfection.

The term “sequence identity” refers to identity between two sequenceswithin a comparison window. Protein sequence identities can be evaluatedusing any of the variety of sequence comparison algorithms and programsknown in the art. For sequence comparison, typically one sequence actsas a reference sequence (e.g., a sequence disclosed herein), to whichtest sequences are compared. A sequence comparison algorithm thencalculates the percent sequence identities for the test sequencesrelative to the reference sequence, based on the program parameters.

The percent identity of two amino acid sequences can be determined forexample by comparing sequence information using the computer programGAP, i.e., Genetics Computer Group (GCG; Madison, Wis.) Wisconsinpackage version 10.0 program, GAP (Devereux et al. (1984), Nucleic AcidsRes. 12: 387-95). In calculating percent identity, the sequences beingcompared are typically aligned in a way that gives the largest matchbetween the sequences. The preferred default parameters for the GAPprogram include: (1) The GCG implementation of a unary comparison matrix(containing a value of 1 for identities and 0 for non-identities) fornucleotides, and the weighted amino acid comparison matrix of Gribskovand Burgess, ((1986) Nucleic Acids Res. 14: 6745) as described in Atlasof Polypeptide Sequence and Structure, Schwartz and Dayhoff, eds.,National Biomedical Research Foundation, pp. 353-358 (1979) or othercomparable comparison matrices; (2) a penalty of 8 for each gap and anadditional penalty of 2 for each symbol in each gap for amino acidsequences, or a penalty of 50 for each gap and an additional penalty of3 for each symbol in each gap for nucleotide sequences; (3) no penaltyfor end gaps; and (4) no maximum penalty for long gaps.

Sequence identity and/or similarity can also be determined by using thelocal sequence identity algorithm of Smith and Waterman, 1981, Adv.Appl. Math. 2:482, the sequence identity alignment algorithm ofNeedleman and Wunsch, 1970, J. Mol. Biol. 48:443, the search forsimilarity method of Pearson and Lipman, 1988, Proc. Nat. Acad. Sci.U.S.A. 85:2444, computerized implementations of these algorithms(BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package,Genetics Computer Group, 575 Science Dr., Madison, Wis.).

Another example of a useful algorithm is PILEUP. PILEUP creates amultiple sequence alignment from a group of related sequences usingprogressive, pairwise alignments. It can also plot a tree showing theclustering relationships used to create the alignment. PILEUP uses asimplification of the progressive alignment method of Feng & Doolittle,1987, J. Mol. Evol. 35:351-360; the method is similar to that describedby Higgins and Sharp, 1989, CABIOS 5:151-153. Useful PILEUP parametersincluding a default gap weight of 3.00, a default gap length weight of0.10, and weighted end gaps.

Another example of a useful algorithm is the BLAST algorithm, describedin: Altschul et al., 1990, J. Mol. Biol. 215:403-410; Altschul et al.,1997, Nucleic Acids Res. 25:3389-3402; and Karin et al., 1993, Proc.Natl. Acad. Sci. U.S.A. 90:5873-5787. A particularly useful BLASTprogram is the WU-BLAST-2 program obtained from Altschul et al., 1996,Methods in Enzymology 266:460-480. WU-BLAST-2 uses several searchparameters, most of which are set to the default values. The adjustableparameters are set with the following values: overlap span=1, overlapfraction=0.125, word threshold (T)=II. The HSP S and HSP S2 parametersare dynamic values and are established by the program itself dependingupon the composition of the particular sequence and composition of theparticular database against which the sequence of interest is beingsearched; however, the values may be adjusted to increase sensitivity.

An additional useful algorithm is gapped BLAST as reported by Altschulet al., 1993, Nucl. Acids Res. 25:3389-3402. Gapped BLAST uses BLOSUM-62substitution scores; threshold T parameter set to 9; the two-hit methodto trigger ungapped extensions, charges gap lengths of k a cost of 10+k;X_(u) set to 16, and X_(g) set to 40 for database search stage and to 67for the output stage of the algorithms. Gapped alignments are triggeredby a score corresponding to about 22 bits.

The term “subject” refers to organisms susceptible to being infectedwith a given coronavirus and may be represented by different species ofbirds and mammals, including, without limitations, humans and non-humanmammals. Thus, subjects susceptible to avian infectious bronchitisinclude poultry, subjects susceptible to Porcine Epidemic Diarrheainclude swine, and subjects susceptible to SARS CoV-2 include cats,dogs, Mustellidae (ferrets, sabers, minks, weasels) and humans.

The term “treating” refers to reducing or alleviating magnitude and/orduration of at least one symptom of an existing coronavirus infection.

The term “vaccine” refers to an immunogenic composition that elicitsprotective immune response in the subject. when administered to asubject, induces or stimulates a protective immune response. A vaccinecan render an organism immune to a particular disease, in the presentcase coronavirus infection, and more particularly SARS-CoV-2 infection.The vaccine of the present invention thus induces an immune response ina subject which is protective against subsequent SARS-CoV-2 challenge. Avaccine comprising the antigen and the adjuvant of the invention may becapable of inducing a cross-protective immune response against aplurality of coronavirus genotypes.

Antigens

The antigen used in the compositions described herein is an inactivatedcoronavirus or Spike protein of the coronavirus or an immunogenicfragment of said Spike protein.

Multiple coronaviruses are suitable for the use in the compositionsdescribed herein. These coronaviruses include, without limitations,Porcine Epidemic Diarrhea Virus (PEDV), Swine Delta Coronavirus (CoV),Feline Infectious Peritonitis Virus, Feline Enteric CoV, AvianInfectious Bronchitis Virus, Turkey CoV, Canine CoV, Canine RespiratoryCoV, Bovine CoV, Equine CoV, TGEV, Porcine Respiratory CoV, PorcineHemagglutinating Encephalomyelitis Virus.

In certain embodiments, the recombinant spike protein antigen comprisesa wild-type 2019-nCoVS protein having the amino acid sequence of SEQ IDNO: 11 or a sequence that is at least 80% identical thereto (e.g., atleast 85% or at least 90% or at least 91% or at least 92% or at least93% or at least 94% or at least 95% or at least 96% or at least 97% orat least 98% or at least 99% or at least 99.5% identical to SEQ ID NO:11), with a proviso that the protein is in a prefusion conformation.Sequence identity should be determined without considering theN-terminal signal peptide “MFVFLVLLPLVSS” (SEQ ID NO: 14).

In certain embodiments, the prefusion conformation is achieved byintroducing mutations between Heptad Repeat 1 and Central Helix of SEQID NO: 11 (or a sequence at least 80% identical thereto as discussedabove). Amino acids at positions 986 and 987 of SEQ ID NO: 11 areparticularly suitable for the mutations. In certain embodiments aminoacids 986 and 987 are both replaced with proline.

In certain embodiments, furin cleavage site PRRARS (SEQ ID NO: 15) thatis generally present between the S1 and S2 domains of the spike proteinis mutated so that furin does not cleave the antigen. In certainembodiments, SEQ ID NO: 15 is mutated into SEQ ID NO: 16 (PGSASS).

In certain embodiments, the recombinant spike protein comprises aC-terminal T4 fibritin foldon motif, such as“GYIPEAPRGDQAYVRKDGEWVLLSTFL” (SEQ ID NO: 12), and, optionally, apurification tag such as a C-terminal polyhistidine tag.

In a preferred embodiment, amino acids of the recombinant spike proteinaccording to the invention corresponding to amino acids at positions 986and 987 of SEQ ID NO: 11 are proline residues, the furin cleavage siteis mutated into SEQ ID NO: 16, the protein comprises the foldon sequenceof SEQ ID NO: 12 and the C-terminal polyhistidine purification tag.

In certain embodiments, the amino acids differing between therecombinant spike protein are conservative substitutions.

In another embodiment, the antigen is a fragment of the wild-type2019-nCoVS protein, as described above, with a proviso that the fragmentcomprises both comprising the S1 and S2 domains.

In certain embodiments, the fragment corresponds to residues 14 to 1208of the wild-type 2019-nCoVS protein of SEQ ID NO: 11, as provided in SEQID NO: 13, or a sequence that is at least 80% identical thereto (e.g.,at least 85% or at least 90% or at least 91% or at least 92% or at least93% or at least 94% or at least 95% or at least 96% or at least 97% orat least 98% or at least 99% or at least 99.5% identical to SEQ ID NO:13), with a proviso that the protein is in a prefusion conformation,and, optionally, with a further proviso that the furin cleavage site ofsaid fragment is non-functional. As described above, the pre-fusionconformation may be achieved by substituting residues 973 and/or 974 ofSEQ ID NO: 13 (corresponding to residues 986 and 987 of SEQ ID NO: 11).The preferred substitution will entail proline residues at bothpositions.

The fragment may further comprise foldon and/or an immunopurificationtag, as described above. In certain embodiments, the fragment of theSpike protein of a coronavirus is a conservatively substituted variantof SEQ ID NO: 13. In the most preferred embodiment, the antigencomprises (or consists of) SEQ ID NO: 17.

Methods of preparing the antigen according to the invention are wellknown. For example, genetic engineering techniques and recombinant DNAexpression systems may be used.

Nucleic acid molecules encoding the amino acid sequences of the antigenaccording to any embodiment of the invention may also be inserted into avector (e.g., a recombinant vector) such as one or more non-viral and/orviral vectors. Non-viral vectors may include, for instance, plasmidvectors (e.g., compatible with bacterial, insect, and/or mammalian hostcells). Exemplary vectors may include, for example, PCR-ii, PCR3, andpcDNA3.1 (Invitrogen, San Diego, Calif.), pBSii (Stratagene, La Jolla,Calif.), pet15 (Novagen, Madison, Wis.), pGEX (Pharmacia Biotech,Piscataway, N.J.), pEGFp-n2 (Clontech, Palo Alto, Calif.), pET1(Bluebacii, Invitrogen), pDSR-alpha (PCT pub. No. WO 90/14363) andpFASTBACdual (Gibco-BRL, Grand island, N.Y.) as well as Bluescriptplasmid derivatives (a high copy number COLe1-based phagemid, StratageneCloning Systems, La Jolla, Calif.), PCR cloning plasmids designed forcloning TAQ-amplified PCR products (e.g., TOPO™ TA Cloning® kit, PCR2.1®plasmid derivatives, Invitrogen, Carlsbad, Calif.). Bacterial vectorsmay also be used including, for instance, Shigella, Vibrio cholerae,Lactobacillus, Bacille Calmette Guerin (BCG), and Streptococcus (see forexample, WO 88/6626; WO 90/0594; WO 91/13157; WO 92/1796; and WO92/21376). The vectors may be constructed using standard recombinanttechniques widely available to one skilled in the art. Many othernon-viral plasmid expression vectors and systems are known in the artand may be used.

Various viral vectors that have been successfully utilized forintroducing a nucleic acid to a host include retrovirus, adenovirus,adeno-associated virus (AAV), herpes virus, baculovirus, and poxvirus,among others. Viral vectors may be constructed using standardrecombinant techniques widely available to one skilled in the art. See,e.g., Molecular cloning: a laboratory manual (Sambrook & Russell: 2000,Cold Spring Harbor Laboratory Press; ISBN: 0879695773), and: Currentprotocols in molecular biology (Ausubel et al., 1988+updates, GreenePublishing Assoc., New York; ISBN: 0471625949). The vectors may be usedto infect host cells, such as, for example, a bacterium, a yeast cell(e.g., a Pichia cell), an insect cell, or a mammalian cell (e.g., CHOcell), and the expressed proteins can be harvested and purifiedaccording to the methods known in the art.

Expression of the amino acid sequences of the antigens recited hereinmay also be performed in so-called cell-free expression systems. Suchsystems comprise all essential factors for expression of the nucleicacid encoding the antigens, operably linked to a promoter that iscapable of expression in that particular system. Examples are the E.coli lysate system (Roche, Basel, Switzerland), or the rabbitreticulocyte lysate system (Promega corp., Madison, USA).

In certain embodiments, SEQ ID NO: 17 is prepared by expressing an aminoacid sequence comprising SEQ ID NO: 17 and the signal peptide of SEQ IDNO: 14 upstream of SEQ ID NO: 17. SEQ ID NO: 14 is cleaved off duringthe processing.

The antigen according to any of the embodiments may be present in theimmunogenic compositions recited herein in the immunologically effectiveamount, sufficient to cause immune response, and preferably, protectiveimmune response. Generally, the immunologically effective amount is theamount of between about 1 μg and 1 mg per dose. In the embodiments wherethe recombinant spike protein or a fragment thereof is the antigen, theprotein or the fragment thereof may be present in the amount of about 1μg to about 500 μg per dose, or between about 1 μg and about 200 μg, orbetween about 2 μg and about 100 μg or between about 5 μg and about 50μg, or between about 10 μg and about 25 μg, or about 20 μg.

Adjuvants

Multiple adjuvanting compounds are known in the art, including, withoutlimitations, saponins, sterols, quaternary ammoniums, glycolipidsstimulating Th2 response, polymers, especially, polymers of apolyacrylic acid, and immunostimulatory oligonucleotides.

Saponins

Suitable saponins include triterpenoid saponins. These triterpenoids agroup of surface-active glycosides of plant origin and share commonchemical core composed of a hydrophilic region (usually several sugarchains) in association with a hydrophobic region of either steroid ortriterpenoid structure. Because of these similarities, the saponinssharing this chemical core are likely to have similar adjuvantingproperties. Triterpenoids suitable for use in the adjuvant compositionscan come from many sources, either plant derived or syntheticequivalents, including but not limited to, Quillaja saponaria, tomatine,ginseng extracts, mushrooms, and an alkaloid glycoside structurallysimilar to steroidal saponins.

If a saponin is used, the adjuvant compositions generally contain animmunologically active saponin fraction from the bark of Quillajasaponaria. The saponin may be, for example, Quil A or another purifiedor partially purified saponin preparation, which can be obtainedcommercially. Thus, saponin extracts can be used as mixtures or purifiedindividual components such as QS-7, QS-10, QS-17, QS-18, and QS-21. Inone embodiment the Quil A is at least 85% pure. In other embodiments,the Quil A is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% pure.

If the triterpenoid saponin (e.g., Quil A or a purified fraction hereof)may be present in the vaccine, then in certain embodiments, one dose ofthe vaccine may contain 1-1000 μg of said triterpenoid saponin, or10-100, or 5-50 or 1-25, or 25-300 or 50-200 or 50-100 μg per dose. Forneonates smaller animals (e.g., dogs or cats or minks), the amount maybe between about 1 and about 100 μg per dose (e.g., between about 5 andabout 50 μg per dose, or between about 10 and about 25 μg per dose, orbetween about 15 and about 20 μg per dose), and for larger animals(e.g., horses, pigs, or cattle) the amount may be between about 50 andabout 1000 μg per dose.

Sterols

Sterols share a common chemical core, which is a steroid ringstructure[s], having a hydroxyl (OH) group, usually attached tocarbon-3. The hydrocarbon chain of the fatty-acid substituent varies inlength, usually from 16 to 20 carbon atoms, and can be saturated orunsaturated. Sterols commonly contain one or more double bonds in thering structure and also a variety of substituents attached to the rings.Sterols and their fatty-acid esters are essentially water-insoluble. Inview of these chemical similarities, it is thus likely that the sterolssharing this chemical core would have similar properties when used inthe vaccine compositions of the instant invention. Sterols are wellknown in the art and can be purchased commercially. For example,cholesterol is disclosed in the Merck Index, 12th Ed., p. 369. Suitablesterols include, without limitations, β-sitosterol, stigmasterol,ergosterol, ergocalciferol, and cholesterol.

If the sterol may be present in the vaccine, then in certainembodiments, one dose of the vaccine may contain 1-1000 μg of saidsterol. In different embodiments, the amount of the sterol is 10-100, or5-50 or 1-25, or 25-300 or 50-200 or 50-100 μg per dose. For neonates orsmaller animals (e.g., dogs or cats or minks), the amount may be betweenabout 1 and about 100 μg per dose (e.g., between about 5 and about 50 μgper dose, or between about 10 and about 25 μg per dose, or between about15 and about 20 μg per dose), and for larger animals (e.g., horses,pigs, or cattle) the amount may be between about 50 and about 1000 μgper dose.

CpG-Containing Immunostimulatory Oligonucleotides

The adjuvant component of the vaccine also comprises animmunostimulatory oligonucleotide. Immunomodulatory oligonucleotidesaccording to the invention comprise CpG (and are also referred to as“CpG containing immunostimulatory oligonucleotides”, “CpGoligonucleotides” or simply “CpGs”). The effect of CpG containingoligonucleotides on the immune system has been known for over 20 years.

Generally, the CpGs suitable for the invention are between 15 and 100bases long, e.g., between 15 and 50 bases long, or between 18 and 40bases long or between 20 and 30 bases long, or 20-24 bases long.

Several classes of CpG have been described, including A-class CpGs,B-class CpGs, C-class CpGs, and P-class CpGs. In certain embodiments,the CpG containing immunostimulatory oligonucleotide is a P-class CpG.P-class CpGs are characterized by the presence of one or more TLR-9activating motif(s) and two palindromes or two complementarity areas.Preferably, the one or more TLR-9 activating motifs are at the 5′ of theoligonucleotide and may be completely or partially be incorporated intothe 5′ palindrome or the 5′ complementarity area. TLR-9 activatingmotifs are known and include, without limitations, TCG, TTCG, TTTCG,TYpR, TTYpR, TTTYpR, UCG, UUCG, UUUCG, TTT, or TTTT. The 5′ palindromeor the 5′ complementary area is at least 6 bases long. The 3′ palindromeor the 3′ complementary area is at least 8 bases long and is generallyrich in C and G. These structural features of the P-class CpGs conferthe ability to spontaneously self-assemble into concatamers either invitro and/or in vivo.

In order to increase lipophilicity of the CpG oligonucleotides, at leastone lipophilic substituted nucleotide analog may be included, preferablyat the 5′ end of the oligonucleotide. The P-class immunostimulatoryoligonucleotides may be modified according to techniques known in theart. For example, J-modification refers to iodo-modified nucleotides.E-modification refers to ethyl-modified nucleotide(s). Thus, E-modifiedP-class immunostimulatory oligonucleotides are P-class immunostimulatoryoligonucleotides, wherein at least one nucleotide (preferably 5′nucleotide) is ethylated. Additional modifications include attachment of6-nitro-benzimidazol, 0-Methylation, modification with proynyl-dU,inosine modification, 2-bromovinyl attachment (preferably to uridine).

The oligonucleotides modified by an addition of a lipophilic moiety aregenerally described in US 20100166780.

In certain embodiments, CpGs according to the invention comprise themodified backbone including, without limitations, phosphorothioatemodifications, halogenations, alkylation (e.g., ethyl- ormethyl-modifications), and phosphodiester modifications.

Suitable non-limiting examples of modified P-class immunostimulatoryoligonucleotides are provided below (“*” refers to a phosphorothioatebond, “-” refers to a phosphodiester bond, “JU” refers to5′-lodo-2′-deoxyuridine and “EU” refers to 5-Ethyl-2′-deoxyuridine).

SEQ ID NO: 1 5′ T*C-G*T*C-G*A*C-G*A*T*C-G*G*C*G*C-G*C*G*C*C*G 3′SEQ ID NO: 2 5′ T*C-G*A*C*G*T*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C*G 3′SEQ ID NO: 3 5′ T*C*G*A*C*G*T*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C*G*T 3′SEQ ID NO: 4 5′ JU*C-G*A*C*G*T*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C*G 3′SEQ ID NO: 5 5′ JU*C-G*A*C*G*T*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C*G* T3′SEQ ID NO: 6 5′ JU*C*G*A*C*G*T*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C*G* T3′SEQ ID NO: 7 5′ EU*C-G*A*C*G*T*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C*G 3′SEQ ID NO: 8 5′ JU*C-G*T*C*G*A*C*G*A*T*C*G*G*C*G*G*C*C*G*C*C*G* T3′SEQ ID NO: 9 5′ JU*C*G*T*C*G*A*C*G*A*T*C*G*G*C*G*G*C*C*G*C*C*G* T3′SEQ ID NO: 10 5′ T*C-G*T*C-G*A*C-G*A*T*C-G*G*C*G*C-G*C*G*C*C*G 3′

In certain embodiments, the CpG oligonucleotide according to theinvention comprises any one of SEQ ID NOs 1-10 or an oligonucleotidecomprising at least 15 consecutive bases of any one of SEQ ID NOs 1-10.In the most preferred embodiment, the vaccine comprises anoligonucleotide comprising at least 15 consecutive bases of SEQ ID NO: 8(e.g., at least 16, at least 17, at least 18, at least 19, at least 20,at least 21, at least 22, or at least 23).

The CpG oligonucleotide may be present in the vaccine in the amount of10-400 μg per dose of the vaccine, or 25-300 or 50-200 or 50-100 μg perdose. For neonates or smaller animals (e.g., dogs or cats or minks), theamount may be between about 0.5 μg and about 70 μg per dose (e.g.,between about 2 μg and about 40 μg per dose or between about 5 μg andabout 30 μg per dose or between about 10 μg and about 25 μg per dose, orabout 20 μg per dose), and for larger animals (e.g., horses, pigs, orcattle) the amount may be between about 50 μg and about 400 μg per dose(e.g., between about 100 μg and about 300 μg per dose, or between about150 μg and about 250 μg per dose).

Polyacrylic Acid Polymers

Polyacrylic acid polymers are also suitable adjuvanting compounds. Forexample, CARBOPOL® polymers are polymers of acrylic acid cross-linkedwith polyalkenyl ethers or divinyl glycol. CARBOPOL® has been used in anumber of vaccines.

The polyacrylic polymer may be present in the vaccine in the amount of 0to about 30% v/v, e.g., about 0.001% v/v to about 25% v/v, of about0.005% v/v to about 15% v/v, of about 0.01% v/v to about 10% v/v, ofabout 0.05% v/v to about 1% v/v, and of about 0.05% v/v to about 0.25%v/v.

Glycolipids

Suitable glycolipids are generally those which activate the Th2response. The glycolipids include, without limitations, thoseencompassed by Formula I and that are generally described in US PatentPublication 20070196384 (Ramasamy et al).

wherein, R¹ is hydrogen, or a saturated alkyl radical having up to 20carbon atoms; X is —CH₂—, —O— or —NH—; R² is hydrogen, or a saturated orunsaturated alkyl radical having up to 20 carbon atoms; R³, R⁴, and R⁵are independently hydrogen, —SO₄ ²⁻, −PO₄ ²⁻, —COC₁₋₁₀ alkyl; R⁶ isL-alanyl, L-alpha-aminobutyl, L-arginyl, L-asparginyl, L-aspartyl,L-cysteinyl, L-glutamyl, L-glycyl, L-histidyl, L-hydroxyprolyl,L-isoleucyl, L-leucyl, L-lysyl, L-methionyl, L-ornithinyl, L-phenyalany,L-prolyl, L-seryl, L-threonyl, L-tyrosyl, L-tryptophanyl, and L-valyl ortheir D-isomers.

Examples of a glycolipid are, without limitation,N-(2-Deoxy-2-L-leucylamino-β-D-glucopyranosyl)-N-octadecyldodecanoylamide(BayR® 1005, or R1005) or a salt (e.g., an acetate) thereof.

If the glycolipid may be present in the vaccine, then in differentembodiments, one dose of the vaccine may contain 0.01 mg to about 10 mgper dose mg of the glycolipid. Thus, for example, the glycolipid may bepresent in the amount of about 0.05 to 2 mg per dose, or about 1 toabout 5 mg per dose or about 4 to about 8 mg per dose or about 5 toabout 10 mg per dose. For neonates or smaller animals (e.g., dogs orcats or minks or weasels, the amount may be between about 0.1 and about1 mg per dose (e.g., about 0.25 to about 0.75 mg per dose, or about 0.2mg per dose to 0.4 mg per dose), and for larger animals (e.g., horses,pigs, or cattle) the amount may be between about 1 and about 10 mg perdose.

Quaternary Ammonium Compounds

Quaternary ammonium compounds are ammonium-based compounds with fourhydrocarbon groups. In practice, hydrocarbon groups are generallylimited to alkyl or aryl groups. In a set of embodiments, the quaternaryammonium compounds are composed of four alkyl chains, two of which areC10-C20 alkyls and the remaining two are C1-C4 alkyls. In certainembodiments, the quaternary ammonium is dimethyldioctadecylammonium(DDA) bromide, chloride or another pharmaceutically acceptable counterion.

If the quaternary ammonium compound is present in the vaccine, one doseof the vaccine may contain the quaternary ammonium in amount of 1-1000μg, or 1-1000 μg, 1-500 μg or 10-100 μg, or 5-50 μg or 1-25 μg, or25-300 μg or 50-200 μg or 50-100 μg. For neonates or smaller animals(e.g., dogs or cats or minks), the amount may be between about 1 mg andabout 100 mg per dose (e.g., between about 5 μg and about 50 μg perdose, or between about 10 μg and about 25 μg per dose, or between about15 μg and about 20 μg per dose), and for larger animals (e.g., horses,pigs, or cattle) the amount may be between about 50 μg and about 1000 μgper dose.

In certain embodiments, the adjuvant generally comprises (or consistsof) a combination of a triterpenoid saponin, a sterol, and aCpG-containing immunostimulatory oligonucleotide. Optionally, theadjuvant may further comprise effective amounts of the quaternaryammonium, the glycolipid and/or the polyacrylic acid polymer such asCARBOPOL®. In certain embodiments, the adjuvant lacks effective amounts(or any detectable amounts) of a quaternary ammonium compound, e.g.,avridine or DDAB, a polyacrylic acid polymer such as, for exampleCARBOPOL® and/or the. In certain embodiments, in one dose of thisadjuvant, Quil A is present in the amount of about 10 μg to about 300μg, cholesterol is present in the amount of between about 10 μg andabout 300 μg, and CpG-containing immunostimulatory oligonucleotide ispresent in the amount of between 10 μg and 250 μg. Preferably, theCpG-containing immunostimulatory oligonucleotide consists of orcomprises SEQ ID NO: 8.

In embodiments particularly suitable for canines, Quil A may be presentin the amount of between 10 μg and 50 μg (preferably between about 15 μgand 25 μg, or at about 20 μg), cholesterol may be present in the amountof between 10 μg and 50 μg (preferably between about 15 μg and 25μg, orat about 20 μg), and the CpG-containing immunostimulatoryoligonucleotide may be present in the amount of between about 10 μg andabout 50 μg (preferably between about 15 μg and about 25 μg, or at about20 μg).

In other embodiments, the adjuvant comprises (or consists of) acombination of the CpG-containing immunostimulatory oligonucleotide andthe glycolipid according to Formula I. In certain embodiments, theadjuvant lacks effective amounts (or any detectable amounts) of aquaternary ammonium compound, e.g., avridine or DDAB, a polyacrylic acidpolymer such as, for example CARBOPOL® and/or a triterpenoid saponin,especially a saponin from Q. Saponaria (including Quil A and fractionsthereof). In certain embodiments, one dose of this adjuvant containsbetween about 15 and about 100 μg of the CpG-containingimmunostimulatory oligonucleotide and between about 100 and about 1000μg per dose (e.g., about 250 to about 750 μg per dose, or about 200 μgper dose to 400 μg per dose) of the glycolipid according to Formula I asdescribed above, which, preferably isN-(2-Deoxy-2-L-leucylamino-β-D-glucopyranosyl)-N-octadecyldodecanoylamideor a salt (e.g., acetate) thereof. Preferably, the CpG-containingimmunostimulatory oligonucleotide consists of or comprises SEQ ID NO: 8.

In yet other embodiments, the adjuvant contains Quil A, Cholesterol,DDAB, and CARBOPOL®. In certain embodiments particularly suitable forfelines and Mustellidae animals, Quil A may be present in the amount ofbetween 10 μg and 50 μg (preferably between about 15 μg and 25 μg, or atabout 20 μg), cholesterol may be present in the amount of between 10 μgand 50 μg (preferably between about 15 μg and 25 μg, or at about 20 μg),DDAB may be present in the amount of between about 5 μg and 20 μg, orbetween about 10 μg and about 15 μg, or about 10 μg, and CARBOPOL® ispresent in the amount of about 0.01% to about 0.1%, or about 0.05% v/v.

Excipients

Other components of the compositions can include pharmaceuticallyacceptable excipients, such as carriers, solvents, and diluents,isotonic agents, buffering agents, stabilizers, preservatives,antibacterial agents, antifungal agents, and the like. Typical carriers,solvents, and diluents include water, saline, dextrose, ethanol,glycerol, oil, and the like. Representative isotonic agents includesodium chloride, dextrose, mannitol, sorbitol, lactose, and the like.Useful stabilizers include gelatin, albumin, and the like. Thecompositions can also contain antibiotics or preservatives, including,for example, gentamicin, merthiolate, or chlorocresol. The variousclasses of antibiotics or preservatives from which to select are wellknown to the skilled artisan.

Methods of Vaccine Administration

The compositions described herein are suitable for induction of immuneresponse against Spike protein of a coronavirus. The compositionsdescribed herein are also suitable for the use as a vaccine, i.e., theadministration of the immunogenic composition disclosed herein leads tothe induction of protective immune response against the coronavirus andthus preventing a subject in need thereof from being infected with saidcoronavirus, or, if said subject still gets infected, for reduction ofthe number and/or severity of the symptoms of said coronavirusinfection.

In certain embodiments, the subject in need of the vaccination is abovine, an ovine, a porcine, an equine, or an avian (e.g., chicken,turkey, geese or ducks) subject. In certain embodiments the subject is acanine, a feline or an animal of Mustellidae family (including minks,ferrets, sables and weasels). In other embodiments, the subject is asimian or a human.

The immunogenic compositions according to the invention may beadministered according to the following regimen: a prime dose followedby the boost (or booster) dose about 14 to about 42 days after the primedose. In different embodiments, the booster dose is administered about14 to about 28 days, or about 21 days after the prime dose. In certainembodiments, this regimen provides at least a six-month duration ofimmunity after the booster dose, and preferably, at least a 12-monthduration of immunity (e.g., 6 month-long, 7 month-long, 8 month-long, 9month-long, 10 month-long, 11 month-long duration of immunity). Thus, incertain embodiments, semi-annual or annual revaccinations areenvisioned.

The immunogenic compositions according to the invention may beformulated for and be administered to the subject by any known routes,including the oral, intranasal, mucosal, topical, transdermal, andparenteral (e.g., intravenous, intraperitoneal, intradermal,subcutaneous or intramuscular). Administration can also be achievedusing needle-free delivery devices. Administration can be achieved usinga combination of routes, e.g., the prime administration using a parentalroute and the boost administration using a mucosal route.

The invention will now be described in the following non-limitingexamples.

EXAMPLES Example 1

The objective of the study is to evaluate the efficacy of a recombinantSARS-CoV-2 trimer spike protein vaccine in dogs via the generation ofantibodies with the ability to neutralize SARS-CoV-2 in vitro. Thevaccine protein is recognized as a target of antibody mediated binding.The protein is similar to that utilized for human SARS and MERSvaccines.

Six- to eleven-month-old male (castrated) and female beagle dogs wereused in this study. Nine of these dogs had previous exposure to CanineParvovirus (orally administered MLV vaccine and CPV2c challenge), CanineParainfluenza Virus (orally administered MLV vaccine and CPIV challengestrain D008), and canine distemper virus (orally administered MLVvaccine). Six of these dogs had previous exposure to canine distempervirus (orally administered MLV vaccine). The dogs were healthy andnegative to SARS-CoV-2 via PCR by oropharyngeal swabs prior to Day 0.

Animals were maintained in an appropriate housing environment to meetUSDA Animal Welfare Regulations (9 Code of Federal Regulations, Chapter1, Subchapter A—Animal Welfare), AAALAC (Association for Assessment andAccreditation of Laboratory Animal Care) and Institutional Animal Careand Use Committee (IACUC) guidelines. The dogs were fed dry foodsuitable for the age and nutritional requirements of the animals,moistened if necessary, and provided ad libitum at least once dailythrough the course of the study. Canned food or non-medicatednutritional supplements were given as needed. Water was available adlibitum at all times.

The dogs were randomly assigned to one of three groups, as provided inTable 1.

TABLE 1 Trt No. of Vaccination Blood End of Group Animals Details DayDose Route Collection Study T01 5 REHYDRAGEL ® ONLY (Control) 0, 21 1.0mL SubQ 0, 21, 42 42 20 μg recombinant Trimer Spike T02 5 protein (SEQID NO: 17) with 1% v/v REHYDRAGEL ® adjuvant, Q.S. with 0.063% PBS (LP),pH = 7.4 T03 5 20 μg recombinant Trimer Spike protein (SEQ ID NO: 17)with adjuvant containing Quil A - 20 μg; Cholesterol - 20 μg; SEQ ID NO:8 - 20 μg per dose), Q.S. with 0.063% PBS (LP), pH = 7.4

Blood for pre-screening (approximately 3.0-6.0 mL) was collected priorto Day 0 for titer screening. Blood was collected in SST tubes from allanimals.

Blood samples (approximately 6.0-12.0 mL or as appropriate forindividual dog weight and blood collection guidelines) for serology werecollected in SST tubes from all animals on Days 0 and 21, either a daybefore vaccination (i.e., day −1 and day 20) or the day of thevaccination but before the vaccination itself. On Day 42 (the end of thestudy) the maximum blood volume was calculated for each animal based onindividual animal weight and IACUC guidelines. Blood was collected inSST tubes from all animals.

All animals were observed once on Day -1, twice on Day 0 (prior to and3-6 hours post-vaccination), once daily on Days 1-5, twice on Day 21(prior to and 3-6 hours post-vaccination), once daily on Days 22-26.Clinical observations were for approximately 30 minutes per session.

Injection site observations were recorded on Study Days 0 (prior tovaccination and 3-6 hours after), 1 through 5 for the first injectionsite (left shoulder). Injection site observations were recorded on StudyDays 21 (prior to vaccination and 3-6 hours after), 22 through 26 (rightshoulder).

The vaccines were well-tolerated by the dogs. No injection site pain orswelling observed during the study. Mild elevations of tympanictemperatures were observed in all study groups post-both vaccinations.No abnormal clinical signs were observed in any animals.

For the measurement of SN titer, a known quantity of the virus wascombined with different dilutions of inactivated sera from the testanimals. SN titer was measured by assessing viability of Vero E6 cellsafter the cells were incubated with the mixture of the virus anddifferent dilution of the sera. See Tan et al., Nat Biotech 38:1073-78(September 2020) and Wang et al., J Immunol. Methods 301:21-30 (2005).

For the determination of ELISA titer, plates were coated with 100μl/well of 250 ng/ml protein (SEQ ID NO: 13) in coating buffer.

Peroxidase conjugated rabbit anti-dog IgG (H+L), polyclonal antibody(Jackson ImmunoResearch # 304-035-003, lot 135618) was used as asecondary antibody and TMB Microwell Peroxidase Substrate DAKO True Blue#1601 was used as the substrate. Sera were initially diluted 1:300followed by 1:3 serial dilutions. Secondary antibody was diluted1:30,000 in PBST (PBS+0.05% (w/v) TWEEN®-20). 100 μl/well of sampleserum dilution was added to the plates, and the plates were incubatedand incubated at room temperature for 60 minutes. Secondary antibody wasdiluted 1:30,000 in dilution buffer, and 100 μl/well of this solutionwas added to the wells, and the reaction proceeded for 30 minutes. Theplates were washed (4× with PBST) after sample incubation and afterincubation with the secondary antibody.

Lateral flow test is a semi-quantitative test. Generally, it is a binarytest to determine whether the animal has or lacks antibodies toSARS-CoV2 by the presence or the absence of the visible band indicatingthe presence of the antibodies. However, but lateral flow device mayalso measure the intensity of the band thus providing asemi-quantitative measure of the amount of the antibodies. Forconvenience, this semi-quantitative measure is referred to as LF titer,or “titer measured by LF” or the like. It should be understood, however,that as applied to the Lateral Flow measurements, the term “titer” isnot a titer, in the strict sense.

Lateral Flow titer was measured by loading the sample and a chase buffercontaining a blocker protein such as BSA, a buffer to maintain pH, Tween20, sodium azide, and polyethylene glycol (PEG) 8000 to the sample wellof a lateral flow device in which they are absorbed by a pad. The sampleand buffer are wicked via capillary action through a deposit ofcolloidal gold conjugated with SEQ ID NO: 13.

The recombinant Spike protein-colloidal gold conjugate was prepared byadding a saturating quantity of protein to the gold and incubated for 10minutes, followed by the addition of a BSA blocker and a stabilizerbuffer including BSA and sucrose.

The antibody-gold complex continues to migrate down the test strip untilit crosses a line of deposited reagent (Protein A or G) to immobilizeantibodies. The cross-linking of the antibody-gold complex to thereagent on this line results in an accumulation of colloidal gold on theline, and a visible red line is formed.

SN titers as well as lateral flow measurements and ELISA for individualanimals are summarized in tables 2, 3, and 4, respectively.

TABLE 2 Animal Treatment SN Day SN Day SN Day ID Group 0 21 42 6591558T01 <32 <32 <32 6591183 T01 <32 <32 <32 6586457 T01 <32 <32 <32 6586384T01 <32 <32 <32 6586279 T01 <32 <32 <32 6591094 T02 <32 <64 >20486590969 T02 <32 272 >2048 6586490 T02 <32 55 563 6586341 T02 <32536 >2048 6586287 T02 <32 <32 1026 6591540 T03 <32 489 >2048 6591442 T03<32 441 >2048 6586422 T03 <32 258 >2048 6586350 T03 <32 538 >20486586295 T03 <32 160 >2048

TABLE 3 Day 0 Day 21 Day 42 Animal Group Visual Titer Visual TiterVisual Titer 6586279 T01 Neg 4469 Neg 3411 Neg 5571 6586384 T01 Neg 7147Neg 11932 Neg 4243 6586457 T01 Neg 3598 Neg 6952 Neg 5706 6591183 T01Neg 5632 Neg 10458 Neg 6727 6591558 T01 Neg 3873 Neg 4010 Neg 78436586287 T02 Neg 2420 Pos 20082 Pos 850922 6586341 T02 Neg 7025 Pos365099 Pos 908487 6586490 T02 Neg 5456 Pos 295387 Pos 749547 6590969 T02Neg 5731 Pos 650084 Pos 964848 6591094 T02 Neg 3604 Pos 132687 Pos775755 6586295 T03 Neg 3230 Pos 558603 Pos 757122 6586350 T03 Neg 2552Pos 695001 Pos 935679 6586422 T03 Neg 3661 Pos 556414 Pos 985040 6591442T03 Neg 8359 Pos 541499 Pos 727335 6591540 T03 Neg 3632 Pos 755504 Pos793830 Titer Titer Titer Animal Group Day 127 Day 155 Day 187 6586279T01 2,763 4,684 6,038 6586384 T01 8,636 8,043 2,793 6586457 T01 9,8045,889 6,162 6591183 T01 4,145 10,615 7,083 6591558 T01 5,717 7,436 3,5066586287 T02 115,359 95,210 82,828 6586341 T02 353,214 264,908 257,6886586490 T02 134,871 81,540 23,998 6590969 T02 395,128 187,301 144,4766591094 T02 254,027 142,923 144,150 6586295 T03 467,165 475,987 474,3296586350 T03 361,288 419,237 548,015 6586422 T03 363,699 424,946 649,2386591442 T03 351,571 405,438 446,510 6591540 T03 463,024 437,149 495,005

All animals from group T01 were negative and all animals from groups T02and T03 were positive on days 127, 155, and 187 by visual observation.

TABLE 4 ELISA ELISA ELISA ELISA ELISA ELISA ELISA Animal Group Day 0 Day21 Day 42 Day 99 Day 127 Day 155 Day 187 6586279 T01 300 100 <1000 <300<300 300 300 6586384 T01 100 100 <1000 300 300 300 300 6586457 T01 100100 <1000 <300 <300 <300 300 6591183 T01 100 100 <1000 300 300 300 3006591558 T01 100 100 <1000 <300 <300 300 300 6586287 T02 300 8100 810008,100 2,700 900 900 6586341 T02 100 8100 27000 24,300 8,100 2,700 2,7006586490 T02 100 8100 27000 24,300 2,700 900 900 6590969 T02 100 270081000 24,300 8,100 900 900 6591094 T02 100 8100 27000 8,100 2,700 900900 6586295 T03 100 8100 243000 24,300 24,300 8,100 8,100 6586350 T03100 8100 81000 24,300 24,300 8,100 8,100 6586422 T03 100 8100 24300024,300 8,100 8,100 8,100 6591442 T03 100 8100 243000 72,900 8,100 8,1008,100 6591540 T03 100 24300 243000 24,300 8,100 8,100 8,100

No control animals seroconverted, as expected. In contrast each of theanimals in group T02 and T03 seroconverted. All animals in groups T02and T03 had protective titer on day 42.

Example 2

The objective of the study is to evaluate the efficacy of a recombinantSARS-CoV-2 trimer spike protein vaccine in cats via the generation ofantibodies with the ability to neutralize SARS-CoV-2 in vitro.

Healthy domestic short hair cats of approximately ten months of age wereused in the study. The cats were acclimatized for at least 14 days priorto use in the study.

All animals in the study were healthy prior to Day 0. All animals in thestudy were negative to SARS-CoV-2 by PCR via nasal swab and serologyprior to Day 0. The animals were maintained in an appropriate housingenvironment to meet USDA Animal Welfare Regulations. Environmentalconditions and floor space were consistent with the standard practicesof the testing facility. The animas were fed with a diet according totheir age requirement and provided with water ad libitum.

The randomization was produced using a SAS (SAS release 9.4 or higher,SAS Institute, Cary, N.C.) program developed specifically for the studywith the ranuni function used to generate random numbers.

The animals were treated as summarized in Table 5 below.

TABLE 5 Trt No. of Vaccination Blood End of Group Animals Details DayDose Route Collection Study T01 5 Control: Adjuvant only, as in T02 0,21 1.0 mL SubQ 0, 21, 42 42 T02 5 Per dose: 20 μg recombinant TrimerSpike protein (SEQ ID NO: 17) adjuvant containing 20 μg Quil A, 20 μgCholesterol, 10 μg DDA, 0.05% v/v CARBOPOL ®, Q.S. with DMEM PBS, pH =7.5 T03 5 Per dose: 20 μg recombinant Trimer Spike protein (SEQ ID NO:17) with adjuvant containing 250 μg BAY1005 ® acetate, 50 μg SEQ ID NO:8, Q.S. with DMEM PBS, pH = 6.8)

The animals were observed, and tympanic temperature was measured twicedaily on day 0 (prior to and 3-6 hours post-vaccination), once daily ondays -1, 1-5, twice on day 21 (prior to and 3-6 hours after thevaccination, once daily on days 22-26. Clinical observations were forapproximately 30 minutes per session.

Injection site reactions were observed daily for 5 days after eachvaccination (days 1-5 and 22-26) or until the reactions were no longervisible for call cats.

The vaccines were well-tolerated. No injection site pain or swellingwere observed while on study. No abnormal clinical observations orelevated temperatures were observed while on study.

Antibody responses to the vaccination were measure by the measurement ofserum neutralizing titer, by lateral flow assay and by ELISA.

SN titers and LF titers were measured as described in Example 1.

ELISA Titers were measured as described in Example 1 except in thisstudy, the starting dilution was 1:100 (rather than 1:300) and thesecondary antibody was diluted 1:40,000 rather than 1:30,000. Theresults for the individual animals are provided in tables 6 (SN titer),7 (Lateral Flow), and 8 (ELISA).

TABLE 6 Animal Group SN, Day 0 SN Day 21 SN Day 42 M191610 T01 <32 <32<32 M191687 T01 <32 <32 <32 M191814 T01 <32 <32 <32 M191962 T01 <32 <32<64 M192021 T01 <32 <32 <32 M191628 T02 <32 <32 >2048 M191644 T02 <32<32 >2048 M191733 T02 <32 >2048 >2048 M191776 T02 <32 >2048 >2048M191989 T02 <32 1371 >2048 M191725 T03 <32 558 >2048 M191857 T03<32 >2048 >2048 M191920 T03 <32 361 >2048 M192004 T03 <32 550.5 >2048M191602 T03 <32 >2048 >2048

TABLE 7 Day 0 Day 21 Day 42 Day 181 Day 265 Animal Group Visual TiterVisual Titer Visual Titer Titer Titer M191610 T01 Neg 8973 Neg 9288 Neg10309 18580 N/A M191687 T01 Neg 9545 Neg 10443 Neg 11800 16804 N/AM191814 T01 Neg 9169 Neg 5292 Neg 7804 13916 N/A M191962 T01 Neg 11614Neg 7924 Neg 17164 9359 N/A M192021 T01 Neg 5862 Neg 14558 Neg 1285519127 N/A M191628 T02 Neg 7526 Pos 188166 Pos 521160 171951 128861M191644 T02 Neg 10431 Pos 293915 Pos 342056 245437 171481 M191733 T02Neg 10775 Pos 221439 Pos 401849 186440 179934 M191776 T02 Neg 14205 Pos247635 Pos 462343 314616 288543 M191989 T02 Neg 5853 Pos 226459 Pos411664 267826 208967 M191725 T03 Neg 9878 Pos 142507 Pos 375131 534069626340 M191857 T03 Neg 14056 Pos 243948 Pos 252345 287385 304955 M191920T03 Neg 6730 Pos 164120 Pos 438883 412096 440451 M192004 T03 Neg 11400Pos 157025 Pos 540735 437773 494968 M191602 T03 Neg 7066 Pos 20709 Pos363478 398709 623569 Day 419 Animal Group Visual Titer M191610 T01 N/AN/A M191687 T01 N/A N/A M191814 T01 N/A N/A M191962 T01 N/A N/A M192021T01 N/A N/A M191628 T02 Pos 72,433 M191644 T02 Pos 73,167 M191733 T02Pos 138,427 M191776 T02 Pos 149,226 M191989 T02 Pos 159,924 M191725 T03Pos 263,788 M191857 T03 Pos 374,334 M191920 T03 Pos 153,351 M192004 T03Pos 188,604 M191602 T03 Pos 383,614 N/A - Sample not analyzed

All animals from group T01 were negative and all animals from groups T02and T03 were positive on days 181 and 265 by visual observation.

TABLE 8 ELISA ELISA ELISA ELISA ELISA ELISA ELISA ELISA Animal Group Day0 Day 21 Day 42 Day 86 Day 115 Day 148 Day 181 Day 265 M191610 T01 900300 300 300 900 900 900 N/A M191687 T01 100 100 100 <300 <300 <300 <300N/A M191814 T01 900 300 300 300 900 300 300 N/A M191962 T01 300 300 300300 300 <300 300 N/A M192021 T01 100 100 100 <300 300 <300 <300 N/AM191628 T02 300 24300 72900 72900 72900 72900 72900 24300 M191644 T02300 24300 72900 72900 24300 24300 24300 24300 M191733 T02 900 7290072900 72900 24300 24300 24300 24300 M191776 T02 100 24300 72900 7290024300 24300 24300 24300 M191989 T02 300 24300 72900 72900 72900 7290024300 24300 M191725 T03 900 8100 72900 72900 72900 72900 72900 72900M191857 T03 300 24300 >218700 218700 72900 72900 72900 72900 M191920 T03300 24300 >218700 218700 72900 72900 72900 72900 M192004 T03 3008100 >218700 72900 72900 72900 72900 72900 M191602 T03 300 8100 >218700218700 72900 218700 72900 72900 ELISA ELISA ELISA ELISA ELISA ELISAAnimal Group D289 D300 D328 D356 D384 D419 M191610 T01 N/A N/A N/A N/AN/A N/A M191687 T01 N/A N/A N/A N/A N/A N/A M191814 T01 N/A N/A N/A N/AN/A N/A M191962 T01 N/A N/A N/A N/A N/A N/A M192021 T01 N/A N/A N/A N/AN/A N/A M191628 T02 24,300 24,300 24,300 24,300 24,300 24,300 M191644T02 24,300 24,300 24,300 8,100 8,100 8,100 M191733 T02 24,300 24,30024,300 24,300 24,300 24,300 M191776 T02 24,300 24,300 8,100 8,100 8,10024,300 M191989 T02 24,300 24,300 24,300 24,300 8,100 24,300 M191725 T0372,900 72,900 72,900 72,900 72,900 72,900 M191857 T03 72,900 24,30024,300 24,300 24,300 72,900 M191920 T03 72,900 72,900 72,900 72,90072,900 72,900 M192004 T03 72,900 72,900 72,900 72,900 24,300 24,300M191602 T03 72,900 72,900 72,900 72,900 72,900 72,900 N/A - Sample notanalyzed

These data demonstrate that the vaccines according to the inventioncause robust immune response against COVID-19 spike protein and that theimmune response persists for at least 265 days or more, e.g., twelvemonths or more, or thirteen months or more, or 419 days.

All publications cited in the specification, both patent publicationsand non-patent publications, are indicative of the level of skill ofthose skilled in the art to which this invention pertains. All thesepublications are herein fully incorporated by reference to the sameextent as if each individual publication were specifically andindividually indicated as being incorporated by reference.

1. A composition comprising a coronavirus, a Spike protein of saidcoronavirus or an immunogenic fragment of said Spike protein, and anadjuvant comprising a saponin, a sterol, and a CpG-containingimmunostimulatory oligonucleotide.
 2. The composition according to claim1, wherein said composition is essentially free of quaternary ammoniumcompounds and essentially free of a glycolipid according to Formula I:

wherein, R¹ is hydrogen, or a saturated alkyl radical having up to 20carbon atoms; X is —CH₂—, —O— or —NH—; R² is hydrogen, or a saturated orunsaturated alkyl radical having up to 20 carbon atoms; R³, R⁴, and R⁵are independently hydrogen, —SO₄ ²⁻, —PO₄ ²⁻, —COC₁₋₁₀ alkyl; R⁶ isL-alanyl, L-alpha-aminobutyl, L-arginyl, L-asparginyl, L-aspartyl,L-cysteinyl, L-glutamyl, L-glycyl, L-histidyl, L-hydroxyprolyl,L-isoleucyl, L-leucyl, L-lysyl, L-methionyl, L-ornithinyl, L-phenyalany,L-prolyl, L-seryl, L-threonyl, L-tyrosyl, L-tryptophanyl, and L-valyl ortheir D-isomers.
 3. The composition of claim 1, wherein the adjuvantconsists of the saponin, the sterol, and the CpG-containingimmunostimulatory oligonucleotide.
 4. The composition according to claim1, wherein the Saponin is Quil A and the sterol is selected from thegroup consisting of β-sitosterol, stigmasterol, ergosterol,ergocalciferol, and cholesterol.
 5. The composition according to claim4, wherein the saponin is present in the amount of about 20 μg per doseand the sterol is present in the amount of about 20 μg per dose.
 6. Acomposition comprising a coronavirus, a Spike protein from saidcoronavirus or an immunogenic fragment of said Spike protein, and anadjuvant comprising a CpG containing immunostimulatory oligonucleotideand a glycolipid according to Formula I,

wherein, R¹ is hydrogen, or a saturated alkyl radical having up to 20carbon atoms; X is —CH₂—, —O— or —NH—; R² is hydrogen, or a saturated orunsaturated alkyl radical having up to 20 carbon atoms; R³, R⁴, and R⁵are independently hydrogen, —SO₄ ²⁻, —PO₄ ²⁻, —COC₁₋₁₀alkyl; R⁶ isL-alanyl, L-alpha-aminobutyl, L-arginyl, L-asparginyl, L-aspartyl,L-cysteinyl, L-glutamyl, L-glycyl, L-histidyl, L-hydroxyprolyl,L-isoleucyl, L-leucyl, L-lysyl, L-methionyl, L-ornithinyl, L-phenyalany,L-prolyl, L-seryl, L-threonyl, L-tyrosyl, L-tryptophanyl, and L-valyl ortheir D-isomers.
 7. The composition according to claim 6, wherein theglycolipid isN-(2-Deoxy-2-L-leucylamino-β-D-glucopyranosyl)-N-octadecyldodecanoylamideor a salt thereof.
 8. The composition according to claim 7 wherein thesalt is an acetate.
 9. The composition according to claim 8, whereinsaid composition is essentially saponin-free.
 10. The compositionaccording to claim 6, wherein the composition is essentially free ofquaternary ammonium compounds.
 11. The composition according to claim 6,wherein the adjuvant consists of the glycolipid and the CpG-containingimmunostimulatory oligonucleotide.
 12. The composition according toclaim 6, wherein the glycolipid is present in the amount of about 250 μgper dose.
 13. The composition according to claim 6, wherein theimmunostimulatory oligonucleotide is a P-class immunostimulatoryoligonucleotide characterized by the presence of one or more TLR-9activating motif (s) and two palindromes or two complementarity areas.14. The composition according to claim 13 wherein said P-classimmunostimulatory oligonucleotide is 5′ modified.
 15. The compositionaccording to claim 14, wherein said P class immunostimulatoryoligonucleotide comprises at least 22 contiguous nucleotides of SEQ IDNO:
 8. 16. The composition according to claim 1, wherein the CpGcontaining immunostimulatory oligonucleotide is present in the amount ofabout 20 to about 50 μg per dose.
 17. A composition comprising acoronavirus, a Spike protein from said coronavirus or an immunogenicfragment of said Spike protein, and an adjuvant comprising a saponin, asterol, a quaternary ammonium compound, and a polyacrylic acid polymer.18. The composition according to claim 17, wherein the Saponin is Quil Aand the sterol is selected from the group consisting of β-sitosterol,stigmasterol, ergosterol, ergocalciferol, and cholesterol, and thequaternary ammonium compound is DDAB.
 19. The composition according toclaim 18, wherein said Quil A is present in the amount of about 20 μgper dose, the sterol is cholesterol and is present in the amount ofabout 20 μg per dose, the DDAB is present in the amount of about 10 μgper dose, and the polyacrylic acid polymer is present in the amount ofabout 0.05% v/v.
 20. The composition according to claim 1, wherein thecoronavirus is SARS-2 coronavirus and the antigen is the Spike proteinor the immunogenic fragment thereof.
 21. The composition according toclaim 20, wherein the Spike protein is at least 90% identical to SEQ IDNO: 13, with a proviso that said protein is in a pre-fusion state. 22.The composition according to claim 21, wherein amino acids at positions973 and 974 are substituted with proline residues.
 23. The compositionaccording to claim 20, comprising a mutation in SEQ ID NO:
 15. 24. Thecomposition according to claim 23, wherein SEQ ID NO: 15 is replacedwith SEQ ID NO:
 16. 25. The composition according to claim 20, whereinsaid Spike protein or the immunogenic fragment thereof further comprisesSEQ ID NO:
 12. 26. The composition according to claim 20, wherein theSpike protein comprises SEQ ID NO: 17 or a sequence at least 99%identical thereto).
 27. The composition according to claim 1, whereinsaid a Spike protein of said coronavirus or an immunogenic fragment ofsaid Spike protein is present in the amount of about 20 μg per dose. 28.A method of inducing an immune response in a subject in need thereof,the method comprising administering to said subject the compositionaccording to claim
 1. 29. The method according to claim 28 wherein saidsubject is a canine.
 30. The method according to claim 28 wherein saidimmunogenic composition is administered to said subject in a primeadministration and in a boost administration, wherein the boostadministration is between about 14 and about 42 days after the primeadministration.
 31. The method according to claim 28 wherein said immuneresponse is a protective immune response.
 32. The method according toclaim 31 wherein said protective immune response is retained for atleast six months after the boost administration.
 33. The methodaccording to claim 32 wherein said protective immune response isretained for at least 12 months after the boost administration.